The steroid/thyroid hormone receptors form a superfamily of ligand-dependent transcription factors that influence cell function and fate in eukaryotes. It is known that these receptors transduce extracellular hormonal signals to target genes that contain specific enhancer sequences referred to as hormone-response elements (HREs) (Evans 1988; Green and Chambon 1988). It is also known that each receptor recognizes its own HREs, thus assuring that a distinct response is triggered by different hormones.
Sequence comparisons and mutational analyses of glucocorticoid receptor (GR) have identified functional domains responsible for transcriptional activation and repression, nuclear localization, DNA binding, and hormone binding (Gigure, et al., 1986; Hollenberg, et al., 1987; Rusconi, et al., 1987; Picard and Yamamoto, 1987; Hollenberg and Evans, 1988; Oro, et al., 1988a). The DNA binding domain, which is required to activate transcription, consists of 66-8 amino acids of which about 20 sites, including nine cysteines (C.sub.1 to C.sub.9), are invariant among different receptors (FIG. 1A). The modular structure of members of this receptor superfamily allows the exchange of one domain for another to create functional chimera. This strategy was used to demonstrate that the DNA binding domain is solely responsible for the specific recognition of the HRE in vivo (Green and Chambon, 1987; Giguere, et al., 1987; Petkovich, et al., 1987; Kumar, et al., 1987; Umesono, et al., 1988; Thompson, et al., 1989) and in vitro (Kumar and Chambon, 1988).
By analogy with the proposed structure for Xenopus transcription factor TFIIIA (Miller, et al., 1985), the invariant cysteines are thought to form two "zinc fingers" for specific DNA binding. In a polypeptide encompassing the DNA binding domain of rat GR, it has been shown that each of two Zn(II) is coordinated in a tetrahedral arrangement by four cysteines (Freedman, et al., 1988). Involvement of these cysteines in Zn(II) coordination is also supported by the fact that eight out of nine cysteines, enough to chelate two Zn(II), are required for the receptor function revealed by point mutagenesis experiments (Hollenberg, et al., 1988; Severne, et al., 1988).
Functional models have been proposed in an attempt to coordinate research results with gene regulation mechanisms that function in vivo. As those skilled in the art will know, one such model for the DNA binding domain is "zinc finger model". (A predicted "finger" structure is presented in FIG. 1B; also see Severne, et al., 1988.) In this model, the first four cysteines (C.sub.1 to C.sub.4) chelate one Zn(II) to form Finger 1, which includes a loop of 13 amino acids between C.sub.2 and C.sub.3. Finger 2 is formed by the next four cysteines (C.sub.5 to C.sub.8), since function is retained when the ninth cysteine is changed to an alanine or serine (Severne, et al., 1988). Finger 2 has a loop of 9 amino acids, and is separated by a "Linker" of 15-17 amino acids from Finger 1. Both fingers are functionally required because neither hGR Finger 1 or 2 by itself is sufficient to retain DNA binding and transactivation function (Hollenberg, et al., 1988). This finger model is applicable to all members of the receptor superfamily, indicating a common mode of HRE recognition by a receptor DNA binding domain.
The HREs are structurally related but in fact are functionally distinct. Those for GR (GRE), estrogen receptor (ER) (ERE), and the thyroid receptors (T.sub.3 Rs) (TRE) have been characterized in detail; they consist of a palindromic pair of "half sites" (FIG. 1C) (Evans, 1988; Green and Chambon, 1988). With optimized pseudo- or consensus response elements, only two nucleotides per half site are different in GRE and ERE (Klock, et al., 1987). On the other hand, identical half sites can be seen in ERE and TRE, but their spacing is different (Glass, et al., 1988). Thus, at least two different means are used to achieve HRE diversity.
As the present invention discloses, functional characterization of mutant receptors carrying chimeric DNA binding domains has made it possible to dissect molecular determinants of target gene specificity for GR, ER, and TR. For example, as the present invention discloses, the identity of GR DNA binding domain can be converted into those of ER and TR by changing three and eight amino acids, respectively. The present invention also discloses that a single Gly to Glu change in the first "zinc finger" of the GR receptor produces a receptor with dual HRE (i.e., GRE and ERE) sequence responsiveness. These discoveries localize two structural determinants of target gene specificity and suggest a simple pathway for the co-evolution of receptor DNA binding domains and regulatory networks.
These discoveries also make it possible to convert one receptor into another, and to create engineered receptors that have desired HRE recognition features. They have also enabled the development of assays that are useful for identifying ligands for "orphan" hormone receptors. Such assays are especially advantageous because they eliminate the necessity of constructing chimeric genes and proteins in order to search for ligands that can activate the orphan receptors.